The invention relates to phosphatidyl compounds which contain a defined hydrophilic residue, and to long-circulating liposomes.
Conventional liposomes circulate in the serum for up to 5 hours. However, especially when liposomes are used as a means of drug delivery, it is desirable that they circulate in the bloodstream for as long as possible.
To this end, the so-called xe2x80x9cstealth liposomesxe2x80x9d were developed, which are not destroyed in the bloodstream so quickly. These xe2x80x9cstealth liposomesxe2x80x9d are built up on the basis of phosphatidyl compounds which have an extended polyethylene glycol residue. The polyethylene glycol residue proved to be most effective in producing the desired increase in liposome survival duration when the molecular weight was between 2000 and 3000. A serious disadvantage, however, of these xe2x80x9cstealth-liposomesxe2x80x9d, ie, of these phosphatidyl compounds with a polyethylene glycol residue, is that the compounds are not exactly defined, since the polyethylene glycol residues display different chain lengths.
Maruyama et al. (Int. J. Pharmac. 111 (1994), 103-107) suggested the use of dipalmitoyl phosphatidyl polyglycerols to lengthen the duration of liposome circulation. However, since technical-grade polyglycerols were used as starting material, no uniform products were obtained here either. Technical polyglycerols, which consist of a mixture of polyglycerols with different chain lengths and monoglycerol, and which are characterized by their average molecular weight, were phosphatidylated by means of phospholipase D. The resulting products only led to a small increase in the survival duration of liposomes in the blood.
The object of this invention was thus to provide compounds which increase the survival duration of liposomes and which are of exactly definable composition.
This objective is established according to the invention by means of a compound with the general formula (A) 
where
R1 and R2, independent of each other, stand for hydrogen or a saturated or unsaturated alkyl or acyl residue, which may be branched and/or substituted, R3 stands for hydrogen or an alkyl residue,
n=0 or 1,
x is a whole number from 1 to 4 and
m is a whole number from 2 to 10 if n=0, or a whole number from 1 to 10 if n=1, or 1 if x is greater than 1,
and where, in the case that n=0, the compound is more than 90% uniform with respect to the value of m.
The stepwise synthesisxe2x80x94upon which this invention is basedxe2x80x94of the hydrophilic residues of the phosphatidyl compounds of formula (A) makes it possible to obtain compounds of an exactly defined composition.
The compound of the invention, with the formula (A), is thus not a mixture of various molecules of indefinite composition and chain length, but can be synthesized to have precisely the desired structure. If, for example, the desired product is a triglycerol derivative, ie, x=1 and m=3 in formula (A), the content of monoglycerol, diglycerol, tetraglycerol and higher oligoglycerol derivatives will be low. It is preferable if a glycerol derivative of a certain chain length is obtained that is largely free of glycerol derivatives of other chain lengths. The content of monoglycerol derivatives, in particular, is low, amounting to less than 5%, preferably less than 1% or, even more preferably, less than 0.1% relative to the desired oligoglycerol derivative.
According to the invention, the compound of formula (A) is a uniform compound of defined structure. It is of advantage if, with respect to the value of m, the compound is greater than 95% uniform. It is to greater advantage if it is more than 99% uniform. It is even possible to provide a compound which is more than 99.9% uniform with respect to the value of m.
The compound is preferably an oligoglycerol derivative with 2 to 5 glycerol units, more preferably with 2 to 4 glycerol units. It is to advantage if these are 1.3-linked linear oligoglycerol residues.
According to the invention, the residues R1 and R2, independent of each other, stand for hydrogen, a saturated or unsaturated C1-C24 alkyl or C1-C24 acyl residue, preferably hydrogen or a saturated or unsaturated C8-C24 alkyl or C8-C24 acyl residue, it being beneficial if it at least one of the residues R1 and R2 is an acyl residue.
The residue R3 is preferably hydrogen or an alkyl residue with 1 to 4 carbon atoms.
The compound of formula (A) can be a racemic compound which contains a phospho-rac-(1 or 3)-oligoglycerol linkage, or it can be in the form of a stereospecific isomer. The stereoisomers can have a phospho-sn-1-oligoglycerol linkage or a phospho-sn-3-oligoglycerol linkage. The formation of the stereospecific linkage can be carried out in a manner analogous to those described in the literature (DE 31 30 867 A1; H. Eibl et al., Chem. Phys. Lipids, 28 (1981), 1-5, 41 (1986), 53-63 and 47 (1988, 47-53).
The subject matter of the invention also includes liposomes which contain phospholipids and/or alkyl phospholipids, maybe cholesterol, and 1 to 50 mol % of a compound with the general formula (A), 
or salts thereof, where the cholesterol, the phospholipids, the alkyl phospholipids and the compound of formula (A) together make up 100 mol %, and R1 and R2, independent of each other, stand for hydrogen, a saturated or unsaturated alkyl or acyl residue which may be branched and/or substituted,
R3 stands for hydrogen or an alkyl residue,
n=0 or 1,
x is a whole number from 1 to 4 and
m is a whole number from 2 to 10 if n=0, or a whole number from 1 to 10 if n=1, or 1 if x is greater than 1, and where, in the case that n=0, the compound (A) is more than 90% uniform in respect of the value of m.
The liposomes of the invention have a half-life in serum of up to 18 to 20 hours. Surprisingly, the liposome concentration in blood was found to decrease linearly.
It is beneficial according to the invention if compound (A) displays a uniformity of more than 95% or, even better, more than 99% with respect to the value of m. It is also possible, however, to use compound (A) in practically pure form, ie, more than 99.9% uniform with respect to the value of m.
The liposomes preferably contain a compound of formula (A), in which x=1 and m is a whole number from 2 to 5; it is even more preferable if m is a whole number from 2 to 4.
The residues R1 and R2 of the compound of formula (A) contained in the liposomes can stand, independent of each other, for hydrogen or a saturated or unsaturated C1-C24 alkyl or C1-C24 acyl residue, preferably hydrogen or a saturated or unsaturated C8-C24 alkyl or C8-C24 acyl residue. The substituent is a residue which does not interfere during the preparation. R3 is preferably hydrogen or a C1-C4 alkyl residue.
The compound of formula (A) can be present in the liposomes as a racemic mixture, ie, with a phospho-rac-(1 or 3)-oligoglycerol linkage. It is preferable if it is present in stereospecific form with a phospho-sn-1-oligoglycerol linkage or a phospho-sn-3-oligoglycerol linkage.
It is to advantage if at least one of the residues R1 and R2 of formula (A) is an acyl group.
It is beneficial if liposomes containing the compound of formula (A) with n=0 exhibit an excess negative charge. However, liposomes can also be prepared from compounds of formula (A) in which n=1. In this case, it is better if the liposomes exhibit no excess charge or a positive one.
Besides a compound of formula (A), the liposomes contain phospholipids and/or alkyl phospholipids and maybe cholesterol. It is preferable to use the compound of formula (A) in an amount of 5 to 15 mol %. If the liposomes do not display an excess charge, a composition of 0 to 70 mol % cholesterol, 1 to 50 mol % of a compound of formula (A), and phospholipids and/or alkyl phospholipids is preferred. If there is a negative excess charge, a preferred liposome composition consists of 0 to 70 mol % cholesterol, 1 to 15 mol % of a compound of formula (A), and phospholipids and/or alkyl phospholipids. A higher proportion of compounds of formula (A) with a negative excess charge would lead to instability of the liposomes in the blood circulation. It is to advantage if the liposomes comprise 35 to 43 mol %, in particular 38 to 42 mol % cholesterol, 5 to 15 mol % of a compound of formula (A), and phospholipids and/or alkyl phospholipids.
The phospholipids and/or alkyl phospholipids can, for example, be diacyl phosphoglycerols of defined structure. Generally speaking, these lipid components can be used as compounds of defined structure.
In the case that x greater than 1, it is preferable if the residue xe2x80x94CH2(xe2x80x94CHOH)xxe2x80x94CH2xe2x80x94OH derives from sugar alcohols which have four hydroxyl groups for x=2, five hydroxyl groups for x=3, and 6 hydroxyl groups for x=4. Examples of such residues are mannitol derivatives for x=4, lyxitol derivatives for x=3 and threitol derivatives for x=2.
The liposomes of the invention have a markedly longer half-life in the blood stream. Their half-life is preferably at least 10 hours, better still, more than 12 hours. Half-lives of 18 to 20 hours have been measured for the liposomes of the invention. Surprisingly, the decrease in blood lipid concentration with time was found to be absolutely linear. It is preferable according to the invention if, after 6 hours, more than 50% of the liposomes added are still present in the blood; it is even more preferable if more than 60% are still present.
A particularly surprising property of the liposomes of the invention is their preferred tendency to accumulate in the spleen. Depending on the composition and size of the liposomes, enrichment thereof in the spleen has been found which exceeds enrichment in the liver by a factor of 25. Enrichment in the spleen compared with that in the liver increases with increasing value of m in formula A and with increasing size of the liposomes. With the transition from SUVs (Small Unilamellar Liposomes; diameter about 60 nm) to LUVs (Large Unilamellar Liposomes; diameter about 190 nm), the degree of enrichment in the spleen increases many times over. The preferential accumulation in the spleen also increases as the number of carbon atoms in R1 and R2 increases.
It was found, in addition, that the liposomes of the invention also accumulate in certain tumour tissues. This was observed to be the case, for example, with breast carcinomas induced by nitrosomethylurea (MNU carcinoma).
The liposomes of the invention can also contain one or more pharmaceutical drugs.
Generally speaking, all drugs can be used that can be introduced into the plasma by means of liposomes. Preferred groups of drugs are, on the one hand, cytostatic agents, especially anthracycline antibiotics such as doxorubicin, epirubicin and daunomycin, with doxorubicin being especially preferred. Other preferred anti-tumour drugs are idarubicin, hexadecylphosphocholine, 1-octadecyl-2-methyl-rac-glycero-3-phosphocholine, 5-fluoruracil, cis-platinum complexes such as carboplatin and novantron, and mitomycins.
Other preferred groups of drugs are immunomodulating substances such as citokines, of which interferon and, in particular, xcex1-interferon are given special preference, antimycotic substances (eg, amphotericin B), and drugs to combat protozoan diseases (malaria and trypanosome and leishmania infections). Taxol is another preferred drug.
Yet another group of preferred drugs is the group of lytic drugs, as are described in the DE 41 32 345 A1. The content of this patent application is thus included by way of reference. Preferred drugs are miltefosin, edelfosin, ilmofosin and SRI62-834.
The subject matter of the invention thus includes use of the liposomes according to the invention for preparing an anti-tumour agent, with the drug doxorubicin being given special preference.
The subject matter of the invention also includes use of the liposomes according to the invention for preparing an agent to influence cell proliferation, with the drug preferably being a cytokine, in particular xcex1-interferon.
The subject matter of the invention includes, in addition, a pharmaceutical composition which contains the liposomes described above and, entrapped in the liposomes, one or more pharmaceutical drugs, combined if necessary with standard pharmaceutical diluents, adjuvants, carrier media and fillers.
The liposomes of the invention are prepared using methods which are known per se and with the usual equipment. Typically, a solution containing the various components of the liposome and 1 to 50 mol % of a compound of formula (A) is converted into a lipid suspension which is then pressed under high pressure through nozzles or a perforated disk; the size of the liposomes can be regulated by means of the size of the perforations in the disk. Suitable measures for converting a lipid suspension into liposomes are familiar to persons versed in the art. Preferably, 5 to 15 mol % of a compound of the general formula (A), 35 to 43 mol % cholesterol and 42 to 60 mol % phospholipids and/or alkyl phospholipids are converted into a lipid suspension, which in turn is converted into liposomes by means of suitable measures and in a manner known per se.
These known methods can also be used to make a pharmaceutical formulation which contains the liposomes of the invention and one or more pharmaceutical drugs. To entrap water-insoluble drugs, the drug is dissolved together with the lipid components, while to entrap water-soluble drugs, an aqueous solution which contains the water-soluble drug is added to the lipid film.
The compounds of the invention, having the formula (A), can be prepared in cases where n=1 by linking a defined oligoglycerol with a phosphatidyl ethanolamine by way of the amino group. This results in neutral compounds, ie, compounds without an excess charge. The defined oligoglycerols used for linking are compounds with the formula (B).
In cases where n=0, compounds with the general formula (A) are made by linking a defined oligoglycerol with a phosphatidylglycerol. When n=0, compounds with the general formula (A) can also be madexe2x80x94using a phosphorylation agentxe2x80x94by linking a defined oligogycerol or a C4-C6 sugar alcohol with an alcohol of the formula CH2xe2x80x94OR1xe2x80x94CHOR2xe2x80x94CHOH. As phosphorylation agent, use is made preferably of POCl3.
The preparation of phospholipids from diacyl glycerols is described in the literature (DE 32 29 817 A1; P. Woolley et al., Chem. Phys. Lipids 47 (1988), 55-62; H. Eibl et al., Chem. Phys. Lipids 47 (1988), 63-68), and this method can be applied here.
Using the above-described methods, a racemic compound is formed which contains a phospho-rac-(1 or 3)-oligoglycerol linkage. It is to advantage if stereospecific compounds are formed, which exhibit a phospho-sn-1-oligoglycerol linkage or a phospho-sn-3-oligoglycerol linkage. To make a compound of formula (A), it is preferable to use a linear oligoglycerol of defined chain length.
The subject matter of the invention also includes a protected oligoglycerol of the formula (B), 
where Y is a whole number from 1 to 9 and X is a benzyl, alkyl or tetrahydropropanyl group. It is beneficial if Y is a whole number from 1 to 3. It is possible according to the invention to obtain 1.3-linked oligoglycerols in practically pure form. Oligoglycerols of a predefined chain length can be prepared which contain hardly any impurities in the form of oligoglycerols with different chain lengths. In addition, these oligoglycerols of the invention are practically free of monomeric glycerol. In other words, uniform compounds are obtained, which have a defined structure.
In the oligoglycerol, X can also stand for a different suitable protective group. It is also possible to replace the acetone with another protective group, in particular another ketone.
The invention comprises, in addition, alkyl oligoglycerols of formula (C) 
where Y is a whole number from 0 to 8, preferably a whole number from 1 to 3, and one of the residues X or Z is a saturated or unsaturated alkyl residue and the other residue is hydrogen. These alkyl oligoglycerols are also uniform compounds of defined structure.
The production of oligoglycerols, protected oligoglycerols and alkyl oligoglycerols is of particular interest, because with the help of these starting materials a number of important and novel adjuvants serving as solubilizers and to improve membrane permeation are obtained. Of particular interest with respect to increasing the period for which the liposomes survive in the blood stream is the production of phosphatidyl oligoglycerol derivatives of formula (A), which carry additional hydroxyl groups in the polar area.
Due to the preferred enrichment of the liposomes of the invention in the spleen, these liposomes are suitable generally for the selective introduction of substances into the spleen. These substances may be medicinal products, contrast agents or the like. This is especially important with regard to improving the quality of vaccines, since the spleen plays a major role in the formation of antibodies for the immune system. In the same way, the enrichment of the liposomes according to the invention such as was observed in tumour tissue is of importance with regard to delivering drugs, contrast agents and the like specifically to such tissue.
The following examples, together with the enclosed drawings, explain the invention in more detail. In the drawings: